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INTRODUCTION

The natriuretic peptides comprise at least three ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide [1] (CNP) [2,3]. ANP and BNP are mainly released from the heart [3], whereas CNP is produced in endothelial cells [2,4]. Three distinct natriuretic peptide receptors have been identified and characterized by molecular cloning; these include natriuretic peptide receptor-A (NPRA), natriuretic peptide receptor-B (NPRB) and natriuretic peptide receptor-C (NPRC) [4–7]. ANP and BNP bind to NPRA, a membrane-bound form of guanylyl cyclase, which is termed as guanylyl cyclase-A (GC-A)/NPRA [5,6,8]. CNP binds to NPRB, which is also a membrane guanylyl cyclase and is known as GC-A/NPRB [8]. All three natriuretic peptides bind to NPRC, which lacks guanylyl cyclase activity [5,8]. The ligand binding to NPRA increases the intracellular second-messenger cyclic GMP (cGMP) concentrations by enhanced guanylyl cyclase activity [4,9–12]. The major functions of ANP-NPRA signalling include natriuretic, diuretic, vasodilatory [2,4,11,13,14], antifibrotic and antihypertrophic effects [8,15–17]. The Npr1 (coding for GC-A/NPRA) has been found to lower arterial pressure and to increase guanylyl cyclase activity in a gene dose-dependent manner [13,18]. Previous studies [13,15,18–24] have suggested the important functions of NPRA signalling in cardiac hypertrophy and arterial blood pressure regulation. The interaction of salt sensitivity with ANP/NPRA system in the development of hypertension, cardiac hypertrophy and inflammatory responses is incompletely understood. A high-salt diet contributes to the pathogenesis of hypertension, which is recognized as a multifactorial trait resulting from the effects of a combination of both environmental and genetic factors [25–28]. Disruption of Npr1 gene leads to hypertension and cardiac hypertrophy in null mutant mice [13,15]. Similarly, ANP homozygous null mutant (Nppa−/−) mice also show exaggerated cardiac hypertrophy and elevated blood pressures [15]. Sodium overload has been shown to be linked with essential hypertension, eliciting cardiac remodelling [29–32].

Aldosterone plays an important role in electrolyte transport and exerts direct effects on cardiac hypertrophy and heart failure by binding to mineralocorticoid receptors in the heart and blood vessels [33–35]. In addition, mineralocorticoid receptor antagonism has been shown to reverse myocardial and aortic fibrosis caused by aldosterone [36,37]. In humans, aldosterone has been shown to exert direct adverse effects on the heart that are independent of its effects on arterial pressure [38]. In patients with severe heart failure, the use of a mineralocorticoid receptor antagonist has been found to reduce morbidity and mortality by 30% [38]. Such studies clearly demonstrated the importance of aldosterone in cardiac hypertrophy and arterial blood pressure regulation. ANP inhibits aldosterone synthesis in cultured neonatal rat cardiomyocytes [39] and the adrenal glands [40,41]. The inhibitory effect of ANP on aldosterone synthesis should depend on the functionally active catalytic domain of NPRA [42]. As NPRA signalling counteracts the renin–angiotensin–aldosterone system (RAAS) [43–45], we tested the hypothesis that whether the cardiac angiotensin II (ANG II) and aldosterone levels are increased in Npr1 gene-disrupted mice, but decreased in Npr1 gene-duplicated mice, in a gene dose-dependent manner. We determined the effect of low-salt or high-salt diets on cardiac ANG II, aldosterone and pro-inflammatory cytokine levels in Npr1 gene-targeted (gene-knockout and gene-duplicated) mice having varying gene copy numbers. The part of our ongoing studies has previously reported the adrenal ANG II and aldosterone levels in Npr1 gene-targeted mice [43]. In the present communication, we report the further results of additional findings on the status of cardiac ANG II, aldosterone and pro-inflammatory cytokines in these genetically altered Npr1 mouse models.

MATERIALS AND METHODS

Generation of Npr1 gene-targeted mice

Npr1 gene-targeted (gene-knockout and gene-duplication) mice were generated by homologous recombination as previously described [8,13]. Animals were generated from correctly targeted embryonic stem cells as previously described [46]. F1 gene-knockout heterozygous animals were identified by Southern blot and PCR analysis of DNA extracted from tail biopsies as previously reported [14,15]. Gene-duplicated homozygous and heterozygous mice were identified by Massachusetts Institute of Technology (MIT) markers analysis using primers D3MIT40 and D3MIT101 [47]. All mice were female littermate progenies of the C57/BL6 genetic background. Mice were bred in the Animal Care Facility at the Tulane University Health Sciences Center. The following are the mice genotypes: 1-copy (+/−) is a gene-disrupted heterozygous mice; 2-copy (+/+) is a wild-type mice; 3-copy (++/+) is a gene-duplicated heterozygous mice; and 4-copy (++/++) is a gene-duplicated homozygous mice as previously reported [38]. Animals were maintained in a 12 : 12-h light–dark cycle (0600 to 1800 h) at 25°C. During the 3-week study period, 24- to 28-week-old female mice were given a low-salt (0.05% NaCl), a normal salt (0.3% NaCl) or a high-salt (8% NaCl) diet and tap water ad libitum as previously reported [43]. In the present studies, the survival of animals was as follow: low-salt diet (1-copy, 97%; 2-copy, 97%; 3-copy 100%; 4-copy, 100%), normal salt diet (1-copy, 94%; 2-copy, 91%; 3-copy 97%; 4-copy, 100%) and high-salt diet (1-copy, 84%; 2-copy, 94%; 3-copy 100; 4-copy, 100%). In the present studies, the major limitation was encountered to obtain the sufficient number of Npr1 gene-disrupted homozygous null mutant (−/−; 0-copy) mice. Approximately, 25% Npr1 0-copy null mutant pups die after 1–2 days of birth. It has also been observed that some unborn pups die in utero just before the birth. Above all, approximately, 30% of the adult 0-copy mice die after 6 months of age due to congestive heart failure. Consequently, the adult 0-copy homozygous null mutant mice colonies are significantly reduced as compared with heterozygous (1-copy) and wild-type (2-copy) mice colonies. Thus, due to a low number of adult 0-copy mice colonies, we could not include these animals in the present studies. All protocols were approved by the Institutional Animal Care and Use Committee at Tulane University Health Sciences Center.

Arterial pressure measurement

The arterial pressure of Npr1 mice was measured every other day by the noninvasive computerized tail-cuff method, using a Visitech BP2000 (Visitech Systems Inc., Apex, North Carolina, USA) as previously described [48]. After 7 days of training of the mice for arterial pressure measurement, an average arterial pressure level of five sessions per day was calculated for the analysis.

Urine and urinary sodium and potassium measurements

After the first 3 days adaptation in metabolic cage, mice were provided with a low-salt, a normal salt or a high-salt diet, respectively, for a 3-week periods. Urine was collected and urine volumes were recorded every other day and kept at −20°C until used. Urinary sodium and potassium were measured using Flame Photometer IL973 (Instrumentation Laboratory, Lexington, Massachusetts, USA). Urine outputs, urinary sodium and potassium excretions per day were normalized by kidney weight.

Blood and tissue collection

After mice had been anaesthetized with CO2, blood was collected by cardiac puncture in a tube containing 10 μl 0.25 mol/l EDTA as previously reported [43]. The blood was centrifuged and the separated plasma was kept at −80°C until used. Hearts were removed and blood exuded from them, after which the hearts were weighed, frozen in liquid nitrogen and kept at −80°C until used. The body weight and tibia length of each mouse was measured for normalization of heart weight.

Cardiac angiotensin II assay

Frozen heart tissues were rinsed and homogenized in 10 mmol/l pyrophosphate buffer containing 100 mmol/l NaCl, 1 mmol/l phenylmelthylsulfonylfluoride (PMSF) and 1 mmol/l EDTA with a Polytron (Brinkmann Instruments, Westbury, New York, USA) at a setting of 10 (three times for 30 s) at 4°C as previously described [49]. The homogenate was centrifuged at 40 000g for 40 min at 4°C. The protein concentration of the heart extract was determined using a protein assay reagent (BioRad, Hercules, California, USA) for normalization and assay of cardiac ANG II levels. The extraction and assay of ANG II was performed as previously described [43].

Cardiac aldosterone assay

Frozen heart tissues were rinsed in phosphate buffer and homogenized as described above for ANG II assay. Aldosterone levels in tissue extract and plasma were assayed using a radioimmunoassay kit (Diagnostic System, Webster, Texas, USA) according to the manufacturer's protocol as previously described [21,43]. The assay of aldosterone indicated 100% cross-reactivity; however, corticosterone showed only 0.02% cross-reactivity and all other steroids were undetectable.

Plasma cyclic GMP assay

Blood samples were collected in tubes containing EDTA and immediately centrifuged at 2500 rpm for 10 min at 4°C. The plasma was separated and stored at −80°C until used. The plasma cGMP levels were assayed using a direct cGMP enzyme-linked immunoassay kit (Assay Designs, Ann Arbor, Michigan, USA), according to the manufacturer's protocol.

Assay of pro-inflammatory and cytokines in plasma and heart tissues

The concentration of pro-inflammatory cytokines, including tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β), was measured in plasma and heart tissues by multiplex bead array format (Millipore, Billerica, Massachusetts, USA), using a Bio-Plex instrument (Bio-Rad) according to the manufacturer's guidelines. Spectrally addressed polystyrene beads coated with cytokine-specific monoclonal antibodies were used to capture the cytokines of interest. The instrument sorted out and measured the fluorescent signal from each bead by dual excitation sources.

Statistical analysis

Statistical analyses were performed by two-way analysis of variance (ANOVA) combined with Bonferroni's multiple comparison posthoc test, using the GraphPad PRISM program (version 4.0; GraphPad Software, San Diego, California, USA). Due to the nonnormality of some of the data, we also performed the nonparametric statistical analysis using Kruskal–Wallis test to confirm the ANOVA analysis. The results are presented as mean ± standard error. Significance was set at a P value of less than 0.05.

DISCUSSION

The present findings provide the evidence that cardiac ANG II levels are increased (+) in Npr1 gene-disrupted mice but decreased (-) in Npr1 gene-duplicated mice in a gene dose-dependent manner. As cardiac ANG II plays roles in cardiac remodelling and function [50,51], an increased cardiac ANG II level may participate in the process of cardiac hypertrophy and heart failure in Npr1 null mutant mice. Previous studies have suggested that most of the cardiac ANG II appears to be produced at tissue sites by the conversion of in-situ synthesized ANG I rather than blood-derived ANG I [52]. Factors such as the circulating renin levels and ANG II binding sites in the heart affect cardiac ANG II production [53]. Enzymatic degradation of ANG II and ANG II-type 1 (AT1) receptor-mediated endocytosis also affect the cardiac ANG II levels [53]. Earlier studies have shown that AT1 receptor signalling in cardiac myocytes and fibroblasts elicits growth and fibrosis [54]. It has been reported that ANP inhibits Ang II-stimulated proliferation in foetal cardiomyocytes, indicating the inhibitory role of ANP-NPRA signalling in cardiac hypertrophy [55]. In comparison with a normal salt diet, a high-salt diet increased (+) cardiac ANG II levels only in 1-copy mice, suggesting that an increased (+) cardiac ANG II may promote cardiac hypertrophy in Npr1 gene-disrupted mice. It has also been reported that a high-salt diet causes cardiac hypertrophy in Dahl salt-sensitive rats [56].

Cardiac aldosterone levels are elevated in Npr1 mice fed with a high-salt diet

It has been previously reported that a low-salt diet stimulates plasma aldosterone levels, whereas a high-salt diet suppresses its production [57]. In the present study, a low-salt diet increased (+) plasma aldosterone levels in 1-copy, 2-copy, 3-copy and 4-copy mice compared with mice given a normal salt diet. However, a high-salt diet suppressed (−) plasma aldosterone levels in 1-copy mice, but not in 3-copy and 4-copy mice. It is possible that an increased sodium excretion and urine output with a decreased arterial pressure in 3-copy and 4-copy mice attenuate the inhibitory effect of a high-salt diet on plasma aldosterone levels [58]. Our findings suggest that NPRA signalling exerts protective function with regard to blood volume homeostasis and arterial pressure regulation in Npr1 gene-duplicated mice as compared with Npr1 gene-disrupted mice. Interestingly, it has been reported that a high-salt diet did not affect plasma renin activity in ANP gene-knockout mice as compared with wild-type mice [59]. The authors considered that ANP gene-knockout mice develop a salt-sensitive component of hypertension in association with a failure to downregulate plasma renin activity adequately. There were interactions of salt with Npr1 gene copy numbers for both cardiac and plasma aldosterone levels. However, the data on cardiac ALDO synthesis are still controversial [52,53]. The present results suggest that ANP/NPRA/cGMP signalling reduces the cardiac aldosterone levels and protects the heart from cardiac hypertrophy and remodelling process in disease states.

Effect of salt diets on urine volume and urinary sodium, and potassium levels in Npr1 mice

Variations in dietary sodium chloride intake are closely associated with changes in renal renin and Ang II content [60]. Increased sodium excretion and urine output with relatively lower blood pressure in Npr1 gene-duplicated mice may attenuate the inhibitory effect of a high-salt diet on Ang II levels. ANP-NPRA signalling is critical in mediating the natriuresis and diuresis after acute volume expansion [61,62]. In the present study, urinary sodium excretion and urine output decreased (−) in Npr1 1-copy mice as compared with wild-type mice, but increased (+) in Npr1 gene-duplicated (3-copy and 4-copy) mice with a normal salt, a low-salt or a high-salt diet, respectively. Our result indicates that decreased ANP-NPRA signalling can lead to sodium retention that causes blood pressure elevation in Npr1 gene-disrupted mice. On the contrary, increased (+) ANP-NPRA signalling effectively attenuates sodium retention and blood pressure elevation in Npr1 gene-duplicated mice. These present results further support the concept that ANP-NPRA signalling plays a critical role in mediating natriuresis and diuresis in a gene dose-dependent manner.

In the present study, the plasma and cardiac cytokine levels were decreased (−) in all the groups receiving a low-salt diet compared with mice receiving a normal salt diet, suggesting that salt restriction could reduce the pro-inflammatory cytokine levels. Similar results have also been reported in rats receiving ANG II for 10 days [63]. The present results showed that TNF-α, IL-6 and IL-1α levels were elevated (+) in both plasma and heart tissues of Npr1 mice on a high-salt diet compared with a normal salt diet. Our previous studies have shown that pro-inflammatory cytokines promote ventricular remodelling and contractile dysfunction in Npr1 mice [15]. Although a high-salt diet showed a significant baseline elevation (+) for all cytokines in 1-copy mice, yet only a small increase occurred in 3-copy and 4-copy mice. The data suggest that increasing Npr1 gene dosage may play a regulatory role in maintaining the pro-inflammatory cytokine levels in sodium-overloaded mice.

Role of a high-salt diet on heart weight/tibia length and heart weight/body weight ratios in Npr1 mice

In the present study, the ratios of heart weight/tibia length and heart weight/body weight increased (+) in Npr1 gene-disrupted 1-copy mice compared with wild-type mice, but decreased (−) in Npr1 gene-duplicated 4-copy mice, implicating that NPRA signalling protects the heart. The high-salt diet increased (+) arterial pressure only in 1-copy and 2-copy mice, but not in 3-copy and 4-copy mice. It has been reported that arterial pressure was decreased (−) in 4-copy mice fed a high-salt diet as compared with 4-copy mice kept on a low-salt diet [13]. Those previous findings suggested that the Npr1 gene, similar to the gene coding for ANP, may directly affect the sensitivity of arterial pressure to salt loading [13]. Although our data provide the evidence that NPRA signalling exerts a protective effect on arterial pressure regulation in mice fed a high-salt diet, in another Npr1 gene-knockout mouse model, a minimal or a high-salt diet did not affect systemic arterial pressure [62]. Both of these models focus on the Npr1 gene disruption, but they have used gene-targeting methods that differ in their details [8,13,62]. However, a similar degree of hypertension has been confirmed in both mouse models. The plasma cGMP levels were decreased (−) in 1-copy mice and increased (+) in 3-copy and 4-copy mice compared with 2-copy mice. The low-salt diet suppressed (−) plasma cGMP levels in all three genotypes of Npr1 mice, whereas the high-salt diet increased (+) plasma cGMP levels in all Npr1 mice. The present results suggest that reduced cGMP signalling increases the heart weight/body weight and heart weight/tibia length ratios impacting cardiac remodelling in Npr1 1-copy gene-disrupted mice.

In conclusion, the present results provide the evidence that cardiac ANG II and aldosterone concentrations are increased (+) in Npr1 gene-disrupted heterozygous (1-copy) mice fed a high-salt diet, however, greatly reduced (−) in Npr1 gene-duplicated (3-copy and 4-copy) mice. The urinary sodium excretion and urine output decreased (−) in Npr1 1-copy mice as compared with wild-type 2-copy mice, but increased (+) in Npr1 gene-duplicated mice. The pro-inflammatory cytokines levels are elevated (+) in both plasma and heart tissues of Npr1 mice kept on a high-salt diet. Furthermore, the high-salt diet showed a significant baseline elevation (+) of pro-inflammatory cytokines in 1-copy and 2-copy mice; yet, the magnitudes of elevation (+) were only small in 3-copy and 4-copy mice. The present results demonstrate that a high-salt diet elevated (+) cardiac ANG II, aldosterone and pro-inflammatory cytokines in 1-copy mice; however, Npr1 gene-duplicated mice did not render such elevated (+) effect indicating the potential role of NPRA against salt-loading and remodelling process in a Npr1 gene dose-dependent manner. The low-salt diet suppressed (−) plasma cGMP levels in all three genotypes of Npr1 mice, whereas the high-salt diet increased (+) plasma cGMP levels in these Npr1 gene-targeted mice. The present results suggest that ANP/NPRA/cGMP signalling decreases (−) the cardiac ANG II, aldosterone and pro-inflammatory cytokine levels and protects the heart from salt-loading and cardiac remodelling process in the disease state.

ACKNOWLEDGEMENTS

We thank Ms Gevoni Bolden and Ms Vickie Nguyen for technical assistance and Mrs Kamala Pandey for assistance during the preparation of this manuscript. We gratefully acknowledge the assistance of Dr Sudesh Srivastav, Department of Biostatistics and Bioinformatics, during the statistical analyses of the data. We are indebted to Professor Oliver Smithies for providing us with the initial breeding pairs of Npr1 gene-targeted mice. Our thanks are due to Dr Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory, MD Anderson Cancer Center, and Dr Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine for providing their facilities during our displacement due to Hurricane Katrina.

This study was funded by a grant from the National Institutes of Health (grant no. HL-62147).

Conflicts of interest

Reviewer's Summary Evaluations Referee 1

The present study contributes to better clarify the role of NPRA system in the heart. By using a solid experimental approach previously introduced and characterized by the same Authors, i.e. Npr1 gene-knockout and gene-duplication mice, this study provides demonstration that Npr1 blunts the local Ang II and aldosterone levels, and attenuates the generation of pro-inflammatory cytokines in the heart of sodium overloaded-mice.

Investigation of the mechanisms underlying the cardiac protection exerted by Npr1 gene was limited to the plasma and cardiac pro-inflammatory cytokines. Whether other pathways may contribute to the protective effect remains unexplored.

Referee 2

The strength of this study is the choice of the type of animal model used to demonstrate that the natriuretic peptide receptor-A gene (Npr 1) plays a pivotal role in the regulation of the cardiac angiotensin II and aldosterone system as the cardiac angiotensin II and aldosterone concentrations are reduced in a gene dose-related way in the response to a high salt diet in gene duplicated mice which also accounts for a protective effect of the Npr 1 gene against cardiac hypertrophy.

Also, a further important message in this respect is that the increased levels of inflammatory cytokines (TNF-alpha, IL-6, IL-1-alpha) in response to a high salt diet are reduced in gene duplicated mice. However, the observation that the angiotensin II plasma levels were not reduced in gene duplicated mice even though the cardiac angiotensin II concentrations were reduced (which contrasts to the plasma aldosterone levels) still requires further explanation.

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